The goal of this project is to elucidate the mechanisms underlying the developmental and homeostatic regulation of gene expression in luteinizing hormone releasing hormone (LHRH) and magnocellular oxytocin (OT) and vasopressin (VP) neurons in the hypothalamus, and substance P and calcitonin gene-related peptide (CGRP)-synthesizing sensory neurons in peripheral ganglia. We have used a slice-explant tissue culture system which maintains differentiated LHRH- and OT-neurons for long periods of time in vitro, to study the effects of potassium depolarization and second messenger activation on neuropeptide gene expression. Assays of mRNA levels in single cells was done by quantitative in situ hybridization histochemistry and image analysis. We found that 40 mM K+ increased OT mRNA levels two- fold, but had no observable effect on LHRH mRNA levels. Both cell types, however, responded to this stimulus by increased c-fos expression. Forskolin treatment resulted in an increase in neuropeptide mRNA in both OT and LHRH neurons after an 8 hr exposure. Experiments to evaluate the turnover rates of LHRH and OT mRNA in these cultures utilized actinomycin D to inhibit transcription. The rate of decay of mRNA after this treatment suggested a relatively fast turnover for LHRH mRNA (t1/2 <24 hr), and a much longer turnover rate (t1/2 greater than or equal to 48 hr) for OT mRNA. Studies of the suprachiasmatic nucleus in vitro showed VP expressing cells exhibited developmental changes (increased levels of expression) similar to that seen in vivo. Numerous vasoactive intestinal polypeptide cells were detected in these explants while gastrin releasing polypeptide, known to be robustly coexpressed in these same cells at later postnatal times in vivo showed low level expression independent of culturing time. GABAergic cells comprised a prominent subtype in these explants and electron microscopic analysis revealed many internuclear synapses. We also found that culturing slices on a permeable membrane, Anocell, dramatically increases survival of OT neurons in vitro. Estrogen receptor (E2 R) mRNA probes were validated in uterus and pituitary in vivo, and used to show that OT cells do not contain E2R. We have characterized developmental expression patterns of substance P and CGRP in neurons in the rat trigeminal ganglion (TGG) in vivo, showing that both peptides reach maximal levels of expression in adult animals. In addition, we have begun to examine the enzymes implicated in the posttranslational modification of neuropeptides into their biologically active forms. The content of mRNA encoding carboxypeptidase E, peptidylglycyl-alpha-amidating monooxygenase, prohormone convertase 1 and prohormone convertase 2 was studied. Preliminary results indicate that each of these candidate neuropeptide processing enzymes is expressed in virtually all neurons in the TGG, and does not appear to be selectively expressed in known peptidergic neurons in the ganglion.